The products of the class II HLA genes play a central role in the regulation of the immune response by presenting antigenic peptides (Ag) to helper T cells via the T cell receptor (TcR) or by stimulating non-- self T cells in the absence of added antigen. The former is referred to as the antigen-specific response and the latter as the alloreactive response. HLA-DR, the predominant class II product, is characterized by extensive polymorphism of its beta-chain and virtual invariance of its alpha-chain. T cell responses to HLADR polymorphisms are mediated through the interaction of the TcR with the DR-bound Ag and with external portions of the DR molecule itself. The antigen-specific response is responsible for initiating immune responses to processed antigen and when directed against foreign antigens is beneficial. When directed against self antigens this response is harmful. Alloreactive responses are targeted against foreign DR and are a problem in transplant situations. This proposal will dissect the role of HLA-DR polymorphism in T cell recognition, identifying the important polymorphic residues for both Ag-specific and alloreactive responses. This analysis will be accomplished at two levels, both using antigen presentation systems where DR molecules, either normal or mutated, are the only class II HLA on the cell surface. The first level is clonal, with specific T cell clones being used to identify details of TcR-Ag-DR interaction. The second is at a bulk culture level with an entire array of T cells responding; this models the immune response as it occurs in the individual. T cell stimulation and expansion of certain lineages in the bulk cultures is assayed by quantitating the TcR specific for each lineage. More specifically, a region of the TcR called the 3rd complementarity determining region (CDR3) will be assayed. Currently, the easiest way to identify and quantify CDR3 is to use molecular genetic probes. This proposal will use a new molecular approach to quantify the CDR3 which I call "TcR size spectratyping." The importance of polymorphic amino acids will be determined by the extent of T cell expansion when a mutated DR molecule is used for the stimulation. In this way the role of each polymorphism in the response can be monitored. In parallel to these studies, we will identify those polymorphic amino acid residues involved in the binding of antigenic peptide so that the T cell response can be interpreted as being mediated via peptide binding or through direct TcR interaction. As part of this analysis of peptide binding by DR molecules, we will determine if the conformation of the DR molecule is influenced by the polymorphisms, by the peptide bound, or by the cell used to present the antigen. Determining which of the polymorphic residues play major or minor roles in T cell reactivity can define allowable mismatches in transplantation. This is important in bone marrow transplantation where for some individuals the chance of finding perfect HLA matches is very low. This should also provide a foundation for analysis of responses to infectious agents and define the role of DR polymorphism in autoimmune responses.